Human myosin heavy chain-like proteins

ABSTRACT

The invention provides human myosin heavy chain-like proteins (MHCP) and polynucleotides which identify and encode MHCP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for treating disorders associated with expression of MHCP.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of twohuman myosin heavy-chain-like proteins and to the use of these sequencesin the diagnosis, prevention, and treatment of cell motility,reproductive, immunological, and neoplastic disorders.

BACKGROUND OF THE INVENTION

Cell motility is governed by the interaction between cytoskeletalproteins and other proteins embedded in the cell membranes. Cytoskeletalproteins which partake in the generation of force within the cell aretermed contractile proteins. The energy source of such force generatingactivity is ATP.

Two predominant contractile proteins in all animal cells are actin andmyosin. Actin is present in both soluble and polymerized forms. Forexample, filamentous (polymerized) actin interacts with myosin tocontract or relax muscle tissues, to transport cell organelles throughthe intracellular medium, to cause cell movement, and to separatedaughter nuclei during cytokinesis.

Myosin has a rodlike structure composed of heavy chain and light chainisoforms. Myosin light chains (MLCs) are associated stoichiometricallywith the globular N-terminal domain of myosin heavy chains (MHCs). Theglobular domain also contains the ATP-binding and actin-binding sites.The MHC C-terminal domain structure is predominantly in the form of anα-helical coil, which interacts with the C-terminal domain of a secondMHC monomer to form a coiled coil higher order structure. Heavy-chainisoforms appear to be present in all tissue types studied and mayregulate maximum shortening velocity of the myofibrils and alter thesensitivity of actinomyosin to intracellular calcium ion concentrations.Two MHC isoforms expressed in smooth muscle are derived from alternatesplicing that results in different amino acid sequences at theirnon-helical C-terminal regions. These sequences have been shown tointeract with internal MHC amino acid residues and have a direct effecton crossbridge function and α-helical coiled coil formation (Martin, A.F. et al. (1997) Comp. Biochem. Physiol. B. Biochem. Mol. Biol.117:3-11).

Vertebrate smooth muscle contraction is dependent upon levels of cAMPand intracellular calcium ions ([Ca²⁺ ]_(i)). The sarcoplasmic reticulum(SR) serves as an intracellular store of [Ca²⁺ ]_(i). Followingstimulation by second messenger molecules, such asinositoltrisphosphate, [Ca²⁺ ]_(i) is briefly released from the SR intothe surrounding cytoplasm. [Ca²⁺ ]_(i) binds to calmodulin (CaM), whichactivates CaM-dependent myosin light chain protein kinase (MLCK). MLCKthen phosphorylates MLC. In relaxed muscle, myosin is prevented frominteracting with actin by tropomyosin. Ca²⁺ binds to tropomyosin,causing a conformational change that leads to the release of actin.Phosphorylated MLC interacts with actin, forming actinomyosin, andinitiates the contraction process. Muscle relaxation is brought about byactive transport of Ca²⁺ into the SR by a calcium ATPase pump, and MLCKis inactivated by a cAMP-dependent protein kinase. Interactions betweenthese molecules may be modulated by other proteins. In particular,telokin, a kinase-related protein encoded by the 3' region of thevertebrate smooth muscle MLCK gene, inhibits MLCK-dependentphosphorylation of MLC by modulating the oligomeric state of MLCK andits interaction with dephosphorylated myosin filaments (Nieznanski, K.and Sobieszek, A. (1997) Biochem. J. 322:65-71). Phosphorylation ofcaldesmon by casein kinase II has been shown to regulate theinteractions between caldesmon and smooth muscle myosin and the abilityof caldesmon to cross-link actin and myosin filaments (Sutherland, C. etal. (1994) J. Muscle Res. Cell. Motil. 15:440-456). Elevation ofintracellular cGMP and activation of protein kinase G (PKG) producesrelaxation of smooth muscle. Thirty one potential PKG substrates havebeen identified, including a protein complex containing proteins of 40,33, 28, and 20 kDa (Li, H. et al. (1996) J. Vasc. Res. 33:99-110).

A Caenorhabditis elegans gene which encodes a protein similar to thecoiled coil domain of MHC has recently been identified, but abiochemical or physiological role has yet to be established (Wilson, R.et al. (1994) Nature 368:32-38). Nasmyth, K. and Jansen, R. P. (1997;Curr. Opin. Cell Biol. 9:396-400) have suggested that proteins of thecytoskeleton, including unconventional myosins, play active roles in thesegregation of differentiation factors and mRNA species during oogenesisand cell differentiation.

Numerous pathologies have been associated with mutations encoded withinMHC isoforms, with differential expression of myosin heavy chainisoforms, and with differential activation of enzymes which chemicallymodify myosin or myosin-associated proteins (Abchee, A. and Marian, A.J. (1997) J. Investig. Med. 45:191-196). For example, elevated levels ofPKCβ2 isoform associated with diabetes mellitus increase transcriptionalactivation of the fetal myosin heavy chain gene in adult myocardium.Together with increases in transcriptional activation of other genes,such as atrial natriuretic factor, c-fos, transforming growth factor,and collagens, this may lead to cardiomyopathy (Wakasai, H. et al.(1997) Proc. Natl. Acad. Sci. 94:9320-9325). Stromal nodules in benignprostatic hyperplasia (BPH) have morphological, cytoskeletal, andbiochemical similarities to fetal prostate stroma supporting the idea ofa reactivation of fetal processes in BPH (Bierhoff, E. et al. (1997)Prostate 31:234-240).

The discovery of two new human myosin heavy chain-like proteins and thepolynucleotides encoding them satisfies a need in the art by providingnew compositions which are useful in the diagnosis, prevention andtreatment of cell motility, reproductive, immunological, and neoplasticdisorders.

SUMMARY OF THE INVENTION

The invention features two substantially purified polypeptides, humanmyosin heavy chain-like proteins collectively referred to as MHCP andindividually referred to as MHCP-1 and MHCP-2, having at least one ofthe amino acid sequences selected from the group consisting of SEQ IDNO:1 and SEQ ID NO:3.

The invention further provides an isolated and substantially purifiedpolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or fragments thereof and a compositioncomprising said polynucleotide sequence. The invention also provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence encoding the amino acid sequence SEQ IDNO:1, or fragments of said polynucleotide sequence. The inventionfurther provides a polynucleotide sequence comprising the complement ofthe polynucleotide sequence encoding the amino acid sequence of SEQ IDNO:1, or fragments or variants of said polynucleotide sequence.

The invention also provides an isolated and purified sequence comprisingSEQ ID NO:2 or variants thereof. In addition, the invention provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence of SEQ ID NO:2. The invention also providesa polynucleotide sequence comprising the complement of SEQ ID NO:2, orfragments or variants thereof.

The present invention further provides an expression vector containingat least a fragment of any of the claimed polynucleotide sequences. Inyet another aspect, the expression vector containing the polynucleotidesequence is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof,the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding MHCP-1 under conditions suitable forthe expression of the polypeptide; and b) recovering the polypeptidefrom the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified MHCP-1 having the amino acid sequence of SEQ IDNO:1 in conjunction with a suitable pharmaceutical carrier.

The invention also provides a purified antagonist of the polypeptide ofSEQ ID NO:1. In one aspect the invention provides a purified antibodywhich binds to a polypeptide comprising the amino acid sequence of SEQID NO:1.

Still further, the invention provides a purified agonist of thepolypeptide of SEQ ID NO:1.

The invention also provides a method for treating or preventing a cellmotility disorder comprising administering to a subject in need of suchtreatment an effective amount of a pharmaceutical composition comprisingpurified MHCP-1.

The invention also provides a method for treating or preventing areproductive disorder comprising administering to a subject in need ofsuch treatment an effective amount of an antagonist to MHCP-1.

The invention also provides a method for treating or preventing animmunological disorder comprising administering to a subject in need ofsuch treatment an effective amount of an antagonist to MHCP-1.

The invention also provides a method for treating or preventing aneoplastic disorder comprising administering to a subject in need ofsuch treatment an effective amount of an antagonist to MHCP-1.

The invention also provides a method for detecting a polynucleotidewhich encodes MHCP-1 in a biological sample comprising the steps of: a)hybridizing the complement of the polynucleotide sequence which encodesSEQ ID NO:1 to nucleic acid material of a biological sample, therebyforming a hybridization complex; and b) detecting the hybridizationcomplex, wherein the presence of the complex correlates with thepresence of a polynucleotide encoding MHCP-1 in the biological sample.In one aspect the nucleic acid material of the biological sample isamplified by the polymerase chain reaction prior to hybridization.

The invention further provides an isolated and substantially purifiedpolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO:3 or fragments thereof and a compositioncomprising said polynucleotide sequence. The invention also provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence encoding the amino acid sequence SEQ IDNO:3, or fragments of said polynucleotide sequence. The inventionfurther provides a polynucleotide sequence comprising the complement ofthe polynucleotide sequence encoding the amino acid sequence of SEQ IDNO:3, or fragments or variants of said polynucleotide sequence.

The invention also provides an isolated and purified sequence comprisingSEQ ID NO:4 or variants thereof. In addition, the invention provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence of SEQ ID NO:4. The invention also providesa polynucleotide sequence comprising the complement of SEQ ID NO:4, orfragments or variants thereof.

The present invention further provides an expression vector containingat least a fragment of any of the claimed polynucleotide sequences. Inyet another aspect, the expression vector containing the polynucleotidesequence is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:3 or a fragment thereof,the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding MHCP-2 under conditions suitable forthe expression of the polypeptide; and b) recovering the polypeptidefrom the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified MHCP-2 having the amino acid sequence of SEQ IDNO:3 in conjunction with a suitable pharmaceutical carrier.

The invention also provides a purified antagonist of the polypeptide ofSEQ ID NO:3. In one aspect the invention provides a purified antibodywhich binds to a polypeptide comprising the amino acid sequence of SEQID NO:3.

Still further, the invention provides a purified agonist of thepolypeptide of SEQ ID NO:3.

The invention also provides a method for treating or preventing a cellmotility disorder comprising administering to a subject in need of suchtreatment an effective amount of a pharmaceutical composition comprisingpurified MHCP-2.

The invention also provides a method for treating or preventing areproductive disorder comprising administering to a subject in need ofsuch treatment an effective amount of an antagonist to MHCP-2.

The invention also provides a method for treating or preventing animmunological disorder comprising administering to a subject in need ofsuch treatment an effective amount of an antagonist to MHCP-2.

The invention also provides a method for treating or preventing aneoplastic disorder comprising administering to a subject in need ofsuch treatment an effective amount of an antagonist to MHCP-2.

The invention also provides a method for detecting a polynucleotidewhich encodes MHCP-2 in a biological sample comprising the steps of: a)hybridizing the complement of the polynucleotide sequence which encodesSEQ ID NO:3 to nucleic acid material of a biological sample, therebyforming a hybridization complex; and b) detecting the hybridizationcomplex, wherein the presence of the complex correlates with thepresence of a polynucleotide encoding MHCP-2 in the biological sample.In one aspect the nucleic acid material of the biological sample isamplified by the polymerase chain reaction prior to hybridization.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, 1D, and 1E show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:2) of MHCP-1. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering Co.Ltd. San Bruno, Calif.).

FIGS. 2A, 2B, 2C, 2D, and 2E show the amino acid sequence (SEQ ID NO:3)and nucleic acid sequence (SEQ ID NO:4) of MHCP-2. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering Co.Ltd. San Bruno, Calif.).

FIG. 3 shows the amino acid sequence alignments between MHCP-1 (2220246;SEQ ID NO:1) and Caenorhabditis elegans myosin heavy chain-like protein(GI 532473; SEQ ID NO:5), produced using the multisequence alignmentprogram of LASER GENE software (DNASTAR Inc, Madison Wis.).

FIGS. 4A and 4B show the amino acid sequence alignments between MHCP-2(3440902; SEQ ID NO:3) and Caenorhabditis elegans myosin heavychain-like protein (GI 532473; SEQ ID NO:5), produced using themultisequence alignment program of LASER GENE software (DNASTAR Inc,Madison Wis.).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the prefer redmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

DEFINITIONS

MHCP, as used herein, refers to the amino acid sequences ofsubstantially purified MHCP obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

The term "agonist", as used herein, refers to a molecule which, whenbound to is MHCP, increases or prolongs the duration of the effect ofMHCP. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of MHCP.

An "allele" or "allelic sequence", as used herein, is an alternativeform of the gene encoding MHCP. Alleles may result from at least onemutation in the nucleic acid sequence and may result in altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

"Altered" nucleic acid sequences encoding MHCP, as used herein includethose with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent MHCP. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding MHCP,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding MHCP. The encoded protein may also be "altered" and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent MHCP.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological or immunological activity of MHCP is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

"Amino acid sequence", as used herein refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof, and tonaturally occurring or synthetic molecules. Fragments of MHCP arepreferably about 5 to about 15 amino acids in length and retain thebiological activity or the immunological activity of MHCP. Where "aminoacid sequence" is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, amino acid sequence, and liketerms, are not meant to limit the amino acid sequence to the complete,native amino acid sequence associated with the recited protein molecule.

"Amplification", as used herein refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term "antagonist", as used herein, refers to a molecule which, whenbound to MHCP, decreases the amount or the duration of the effect of thebiological or immunological activity of MHCP. Antagonists may includeproteins, nucleic acids, carbohydrates, antibodies or any othermolecules which decrease the effect of MHCP.

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fa, F(ab')₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind MHCP polypeptidescan be prepared using intact polypeptides or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide oroligopeptide used to immunize an animal can be derived from thetranslation of RNA or synthesized chemically and can be conjugated to acarrier protein, if desired. Commonly used carriers that are chemicallycoupled to peptides include bovine serum albumin and thyroglobulin,keyhole limpet hemocyanin. The coupled peptide is then used to immunizethe animal (e.g., a mouse, a rat, or a rabbit).

The term "antigenic determinant", as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The term "antisense", as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term "antisense strand" is used in reference toa nucleic acid strand that is complementary to the "sense" strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation "negative" is sometimes used in referenceto the antisense strand, and "positive" is sometimes used in referenceto the sense strand.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic MHCP, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence"A-G-T" binds to the complementary sequence "T-C-A". Complementaritybetween two single-stranded molecules may be "partial", in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

A "composition comprising a given polynucleotide sequence", as usedherein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding MHCP or fragments thereof may be employed as hybridizationprobes. The probes may be stored in freeze-dried form and may beassociated with a stabilizing agent such as a carbohydrate. Inhybridizations, the probe may be deployed in an aqueous solutioncontaining salts (e.g., NaCl), detergents (e.g., SDS) and othercomponents (e.g., Denhardt's solution, dry milk, salmon sperm DNA,etc.).

"Consensus", as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, has been extended usingXL-PCR kit (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3'direction and resequenced, or has been assembled from the overlappingsequences of more than one Incyte Clone using a computer program forfragment assembly (e.g., GELVIEW Fragment Assembly system, GCG, Madison,Wis.). Some sequences have been both extended and assembled to producethe consensus sequence.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2 or SEQ ID NO:4 by northern analysis isindicative of the presence of mRNA encoding MHCP in a sample and therebycorrelates with expression of the transcript from the polynucleotideencoding the protein.

A "deletion", as used herein, refers to a change in the amino acid ornucleotide sequence and results in the absence of one or more amino acidresidues or nucleotides.

The term "derivative", as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to MHCP or theencoded MHCP. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

The term "homology", as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term "substantiallyhomologous." The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

Human artificial chromosomes (HACs) are linear microchromosomes whichmay contain DNA sequences of 10K to 10M in size and contain all of theelements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

The term "hybridization", as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

"Microarray" refers to an array of distinct polynucleotides oroligonucleotides synthesized on a substrate, such as paper, nylon orother type of membrane, filter, chip, glass slide, or any other suitablesolid support.

The term "modulate", as used herein, refers to a change in the activityof MHCP. For example, modulation may cause an increase or a decrease inprotein activity, binding characteristics, or any other biological,functional or immunological properties of MHCP.

"Nucleic acid sequence", as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNAof genomic or synthetic origin which may be single- or double-stranded,and represent the sense or antisense strand. "Fragments" are thosenucleic acid sequences which are greater than 60 nucleotides in length,and most preferably includes fragments that are at least 100 nucleotidesor at least 1000 nucleotides, and at least 10,000 nucleotides in length.

The term "oligonucleotide" refers to a nucleic acid sequence of at leastabout 6 nucleotides to about 60 nucleotides, preferably about 15 to 30nucleotides, and more preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or a hybridization assay, or a microarray.As used herein, oligonucleotide is substantially equivalent to the terms"amplimers", "primers", "oligomers", and "probes", as commonly definedin the art.

"Peptide nucleic acid", PNA as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast five nucleotides in length linked to a peptide backbone of aminoacid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from five amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein "comprising atleast a portion of the amino acid sequence of SEQ ID NO:1" encompassesthe full-length MHCP-1 and fragments thereof and a protein "comprisingat least a portion of the amino acid sequence of SEQ ID NO:3"encompasses the full-length MHCP-2 and fragments thereof.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding MHCP, orfragments thereof, or MHCP itself may comprise a bodily fluid, extractfrom a cell, chromosome, organelle, or membrane isolated from a cell, acell, genomic DNA, RNA, or cDNA (in solution or bound to a solidsupport, a tissue, a tissue print, and the like).

The terms "specific binding" or "specifically binding", as used herein,refers to that interaction between a protein or peptide and an agonist,an antibody and an antagonist. The interaction is dependent upon thepresence of a particular structure (i.e., the antigenic determinant orepitope) of the protein recognized by the binding molecule. For example,if an antibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

The terms "stringent conditions" or "stringency", as used herein, referto the conditions for hybridization as defined by the nucleic acid,salt, and temperature. These conditions are well known in the art andmay be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

A "variant" of MHCP, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE software.

THE INVENTION

The invention is based on the discovery of two new human myosin heavychain-like proteins, MHCP (MHCP-1 and MHCP-2), the polynucleotidesencoding MHCP, and the use of these compositions for the diagnosis,prevention, or treatment of cell motility, reproductive, immunological,and neoplastic disorders.

Nucleic acids encoding the MHCP-1 of the present invention were firstidentified in Incyte Clone 2220246 from the lung cDNA library(LUNGNOT18) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:2, was derived from the followingoverlapping and/or extended nucleic acid sequences: Incyte Clones2220246 (LUNGNOT18), 3176371 (UTRSTUT04), 1342768 (COLNTUT03), 158646(ADENINB01), 1401623 (BRAITUT08), 1284678 (COLNNOT16), and 1887979(BLADTUT07).

Nucleic acids encoding the MHCP-2 of the present invention were firstidentified in Incyte Clone 3440902 from the corpus cavemosum cDNAlibrary (PENCNOT06) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:4, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 3440902 (PENCNOT06), 3204018 (PENCNOT02), 2138118 (ENDCNOT01),1352537 and 1402252 (LATRTUT02), 2098065 (BRAITUT02), 1805554(SINTNOT13), and 1474994 (LUNGTUT03).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, 1C, D,and 1E. MHCP-1 is 226 amino acids in length and has two potentialglycosylation sites at residues N-118 and N-218, eight potential caseinkinase II phosphorylation sites at residues S-65, S-104, T-109, S-143,T-150, S-157, T-168, and T-193, and a leucine zipper between residuesL-71 and L-92. As shown in FIG. 3, MHCP-1 has chemical and structuralhomology with Caenorhabditis elegans myosin heavy chain-like protein (GI532473; SEQ ID NO:5). In particular, MHCP-1 and C. elegans myosin heavychain-like protein share 28% identity and one casein kinase IIphosphorylation site. Northern analysis shows the expression of thissequence in various libraries, at least 48% of which are immortalized orcancerous and at least 23% of which involve immune response. Ofparticular note is the expression of MHCP-1 in lung, small intestine andcolon, brain, uterus, ovary, prostate, and penile tissues.

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:3, as shown in FIGS. 2A, 2B, 2C,2D, and 2E. MHCP-2 is 354 amino acids in length and has two potentialglycosylation sites at residues N-245 and N-345, one potential proteinkinase A or G phosphorylation site at residue S-47, eleven potentialcasein kinase II phosphorylation sites at residues S-59, S-66, T-97,T-164, T-184, S-1 93, S-238, S-272, S-277, S-295, and S-320, sixpotential protein kinase C phosphorylation sites at residues T-20, S-42,S-43, S-59, T-97, and S-1 12, and one potential leucine zipper betweenresidues L-199 and L-220. As shown in FIGS. 4A and 4B, MHCP-2 haschemical and structural homology with C. elegans myosin heavy chain-likeprotein (GI 532473; SEQ ID NO:5). In particular, MHCP-2 and C. elegansmyosin heavy chain-like protein share 36% identity, and one caseinkinase II phosphorylation site. Northern analysis shows the expressionof this sequence in various libraries, at least 44% of which areimmortalized or cancerous and at least 30% of which involve immuneresponse. Of particular note is the expression of MHCP-2 in heart, lung,small intestine and colon, pancreas, bladder, breast, prostate, brain,and thyroid tissues, and in hematopoietic and muscle tissues.

The invention also encompasses MHCP variants. A preferred MHCP variantis one having at least 80%, and more preferably at least 90%, amino acidsequence identity to the MHCP amino acid sequence and retaining at leastone biological, immunological, or other functional characteristic oractivity of MHCP. A most preferred MHCP variant is one having at least95% amino acid sequence identity to SEQ ID NO:1 or SEQ ID NO:3.

The invention also encompasses polynucleotides which encode MHCP.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of MHCP can be used to produce recombinant molecules whichexpress MHCP. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A, 1B, 1C, 1D, and 1E. In another embodiment, theinvention encompasses the polynucleotide comprising the nucleic acidsequence of SEQ ID NO:4 as shown in FIGS. 2A, 2B, 2C, 2D, and 2E.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding MHCP, some bearing minimal homology to the nucleotide sequencesof any known and naturally occurring gene, may be produced. Thus, theinvention contemplates each and every possible variation of nucleotidesequence that could be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet genetic code as applied to the nucleotide sequence ofnaturally occurring MHCP, and all such variations are to be consideredas being specifically disclosed.

Although nucleotide sequences which encode MHCP and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring MHCP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding MHCP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding MHCP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or fragmentsthereof, which encode MHCP and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding MHCP or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2 or SEQ ID NO:4, under variousconditions of stringency as taught in Wahl, G. M. and S. L. Berger(1987; Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987; MethodsEnzymol. 152:507-511).

Methods for DNA sequencing which are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE (U.S. Biochemical Corp, Cleveland, Ohio),Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE Amplification Systemmarketed by Gibco/BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (PerkinElmer).

The nucleic acid sequences encoding MHCP may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,"restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 Primer Analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries (Clontech, Palo Alto, Calif.) to walk genomic DNA. Thisprocess avoids the need to screen libraries and is useful in findingintronlexon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5' non-transcribedregulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. GENOTYPER and SEQUENCE NAVIGATOR,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode MHCP may be used in recombinant DNAmolecules to direct expression of MHCP, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressMHCP.

As will be understood by those of skill in the art, it may beadvantageous to produce MHCP-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter MHCP encodingsequences for a variety of reasons, including but not limited to,alterations which modify the cloning, processing, and/or expression ofthe gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding MHCP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of MHCP activity, it may be useful toencode a chimeric MHCP protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the MHCP encoding sequence and theheterologous protein sequence, so that MHCP may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding MHCP may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of MHCP, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of MHCP, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active MHCP, the nucleotide sequencesencoding MHCP or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding MHCP andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding MHCP. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector--enhancers, promoters, 5' and 3'untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORTI plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding MHCP,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for MHCP. For example, when largequantities of MHCP are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding MHCP may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding MHCP may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196).

An insect system may also be used to express MHCP. For example, in onesuch system, Autographa califomica nuclear polyhedrosis virus (AcNPV) isused as a vector to express foreign genes in Spodoptera frugiperda cellsor in Trichoplusia larvae. The sequences encoding MHCP may be clonedinto a non-essential region of the virus, such as the polyhedrin gene,and placed under control of the polyhedrin promoter. Successfulinsertion of MHCP will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which MHCP may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding MHCP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing MHCP in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding MHCP. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding MHCP, its initiation codon, and upstream sequences are insertedinto the appropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including the ATG initiation codon shouldbe provided. Furthermore, the initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons may be of various origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used, such as those described in the literature(Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressMHCP may be transformed using expression vectors which may contain viralorigins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding MHCP isinserted within a marker gene sequence, transformed cells containingsequences encoding MHCP can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding MHCP under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding MHCP and express MHCP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA--DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding MHCP can be detectedby DNA--DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding MHCP. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding MHCP to detect transformantscontaining DNA or RNA encoding MHCP.

A variety of protocols for detecting and measuring the expression ofMHCP, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson MHCP is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding MHCP includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding MHCP, or anyfragments thereof may be cloned into a vector for the production of anmRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland, Ohio).Suitable reporter molecules or labels, which may be used for ease ofdetection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding MHCP may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeMHCP may be designed to contain signal sequences which direct secretionof MHCP through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding MHCP to nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and MHCP may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingMHCP and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying MHCP from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of MHCP may be producedby direct peptide synthesis using solid-phase techniques (Merrifield J.(1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may beperformed using manual techniques or by automation. Automated synthesismay be achieved, for example, using Applied Biosystems 431A PeptideSynthesizer (Perkin Elmer). Various fragments of MHCP may be chemicallysynthesized separately and combined using chemical methods to producethe full length molecule.

THERAPEUTICS

Chemical and structural homology exists between MHCP-1 andCaenorhabditis elegans myosin heavy chain-like protein (GI 532473; SEQID NO:5). In addition, MHCP-1 is expressed in tumor and proliferatingtissues, lung, small intestine and colon, brain, uterus, ovary,prostate, and penile tissues. Therefore, MHCP-1 appears to play a rolein cell motility, reproductive, immunological, and neoplastic disorders.

Chemical and structural homology exists between MHCP-2 andCaenorhabditis elegans myosin heavy chain-like protein (GI 532473; SEQID NO:5). In addition, MHCP-2 is expressed in tumor and proliferatingtissues, heart, lung, small intestine and colon, pancreas, bladder,breast, prostate, brain, and thyroid tissues, and in hematopoietic andmuscle tissues. Therefore, MHCP-2 appears to play a role in cellmotility, reproductive, immunological, and neoplastic disorders.

In one embodiment, MHCP or a fragment or derivative thereof may beadministered to a subject to treat or prevent a cell motility disorder.Such disorders include, but are not limited to, ankylosing spondylitis,Chediak-Higashi syndrome, Duchenne and Becker muscular dystrophy,intrahepatic cholestasis, myocardial hyperplasia, cardiomyopathy, earlyonset periodontitis, cancers such as adenocarcinoma, ovarian carcinoma,and chronic myelogenous leukemia, and bacterial and helminth infections.

In another embodiment, a vector capable of expressing MHCP, or afragment or a derivative thereof, may also be administered to a subjectto treat or prevent a cell motility disorder including, but not limitedto, those described above.

In still another embodiment, an agonist which modulates the activity ofMHCP may also be administered to a subject to treat a cell motilitydisorder including, but not limited to, those described above.

In one embodiment, an antagonist of MHCP may be administered to asubject to prevent or treat a reproductive disorder. Such disorders mayinclude, but are not limited to, disorders of prolactin production;infertility, including tubal disease, ovulatory defects, andendometriosis; disruptions of the estrous cycle, disruptions of themenstrual cycle, polycystic ovary syndrome, ovarian hyperstimulationsyndrome, endometrial and ovarian tumors, autoimmune disorders, ectopicpregnancy, and teratogenesis; cancer of the breast, fibrocystic breastdisease, and galactorrhea; disruptions of spermatogenesis, abnormalsperm physiology, cancer of the testis, cancer of the prostate, benignprostatic hyperplasia, and prostatitis; carcinoma of the male breast;and gynecomastia. In one aspect, an antibody which specifically bindsMHCP may be used directly as an antagonist or indirectly as a targetingor delivery mechanism for bringing a pharmaceutical agent to cells ortissue which express MHCP.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding MHCP may be administered to a subject to treator prevent a reproductive disorder including, but not limited to, thosedescribed above.

In one embodiment, an antagonist of MHCP may be administered to asubject to prevent or treat an immunological disorder. Such disordersmay include, but are not limited to, AIDS, Addison's disease, adultrespiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome,Werner syndrome, and autoimmune thyroiditis; complications of cancer,hemodialysis, and extracorporeal circulation; viral, bacterial, fungal,parasitic, protozoal, and helminthic infections; and trauma. In oneaspect, an antibody which specifically binds MHCP may be used directlyas an antagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express MHCP.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding MHCP may be administered to a subject to treator prevent an immunological disorder including, but not limited to,those described above.

In one embodiment, an antagonist of MHCP may be administered to asubject to prevent or treat a neoplastic disorder. Such disorders mayinclude, but are not limited to, adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancersof the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus. In oneaspect, an antibody which specifically binds MHCP may be used directlyas an antagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express MHCP.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding MHCP may be administered to a subject to treator prevent a neoplastic disorder including, but not limited to, thosedescribed above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of MHCP may be produced using methods which are generallyknown in the art. In particular, purified MHCP may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind MHCP.

Antibodies to MHCP may be generated using methods that are well known inthe art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith MHCP or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to MHCP have an amino acid sequence consisting of atleast five amino acids and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of MHCP amino acids may be fused with those of another proteinsuch as keyhole limpet hemocyanin and antibody produced against thechimeric molecule.

Monoclonal antibodies to MHCP may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.62:109-120).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceMHCP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter,G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for MHCP mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between MHCP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering MHCP epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingMHCP, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, the complement of the polynucleotide encodingMHCP may be used in situations in which it would be desirable to blockthe transcription of the mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding MHCP. Thus,complementary molecules or fragments may be used to modulate MHCPactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligonucleotides orlarger fragments, can be designed from various locations along thecoding or control regions of sequences encoding MHCP.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encoding MHCP.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra).

Genes encoding MHCP can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes MHCP. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5' or regulatory regions of the gene encoding MHCP(signal sequence, promoters, enhancers, and introns). Oligonucleotidesderived from the transcription initiation site, e.g., between positions-10 and +10 from the start site, are preferred. Similarly, inhibitioncan be achieved using "triple helix" base-pairing methodology. Triplehelix pairing is useful because it causes inhibition of the ability ofthe double helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) In: Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). Thecomplementary sequence or antisense molecule may also be designed toblock translation of mRNA by preventing the transcript from binding toribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding MHCP.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding MHCP. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA constitutivelyor inducibly can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections orpolycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of MHCP, antibodies toMHCP, mimetics, agonists, antagonists, or inhibitors of MHCP. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers may also be used for delivery. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of MHCP, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example MHCP or fragments thereof, antibodies of MHCP,agonists, antagonists or inhibitors of MHCP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind MHCP may beused for the diagnosis of conditions or diseases characterized byexpression of MHCP, or in assays to monitor patients being treated withMHCP, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for MHCP includemethods which utilize the antibody and a label to detect MHCP in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuring MHCPare known in the art and provide a basis for diagnosing altered orabnormal levels of MHCP expression. Normal or standard values for MHCPexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toMHCP under conditions suitable for complex formation. The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric means. Quantities of MHCP expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingMHCP may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofMHCP may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of MHCP,and to monitor regulation of MHCP levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding MHCP or closely related molecules, may be used to identifynucleic acid sequences which encode MHCP. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding MHCP, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe MHCP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or SEQ ID NO:4 or from genomic sequence including promoter,enhancer elements, and introns of the naturally occurring MHCP.

Means for producing specific hybridization probes for DNAs encoding MHCPinclude the cloning of nucleic acid sequences encoding MHCP or MHCPderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding MHCP may be used for the diagnosis ofa disorder associated with expression of MHCP. Examples of such adisorder include, but are not limited to, a cell motility disorder suchas ankylosing spondylitis, Chediak-Higashi syndrome, Duchenne and Beckermuscular dystrophy, intrahepatic cholestasis, myocardial hyperplasia,cardiomyopathy, early onset periodontitis, cancers such asadenocarcinoma, ovarian carcinoma, and chronic myelogenous leukemia, andbacterial and helminth infections; a reproductive disorder such asdisorders of prolactin production; infertility, including tubal disease,ovulatory defects, and endometriosis; disruptions of the estrous cycle,disruptions of the menstrual cycle, polycystic ovary syndrome, ovarianhyperstimulation syndrome, endometrial and ovarian tumors, autoimmunedisorders, ectopic pregnancy, and teratogenesis; cancer of the breast,fibrocystic breast disease, and galactorrhea; disruptions ofspermatogenesis, abnormal sperm physiology, cancer of the testis, cancerof the prostate, benign prostatic hyperplasia, and prostatitis;carcinoma of the male breast; and gynecomastia; an immunologicaldisorder such as AIDS, Addison's disease, adult respiratory distresssyndrome, allergies, anemia, asthma, atherosclerosis, bronchitis,cholecystitis, Crohn's disease, ulcerative colitis, atopic dermatitis,dermatomyositis, diabetes mellitus, emphysema, erythema nodosum,atrophic gastritis, glomerulonephritis, gout, Graves' disease,hypereosinophilia, irritable bowel syndrome, lupus erythematosus,multiple sclerosis, myasthenia gravis, myocardial or pericardialinflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis,rheumatoid arthritis, scleroderma, Sjogren's syndrome, Werner syndrome,and autoimmune thyroiditis; complications of cancer, hemodialysis, andextracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections; and trauma; and a neoplasticdisorder such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, teratocarcinoma, and, in particular, cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. The polynucleotidesequences encoding MHCP may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; orin dipstick, pin, ELISA assays or microarrays utilizing fluids ortissues from patient biopsies to detect altered MHCP expression. Suchqualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding MHCP may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingMHCP may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding MHCP in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of MHCP, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes MHCP, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding MHCP may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5'→3') and another with antisense (3'←5'),employed under optimized conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

Methods which may also be used to quantitate the expression of MHCPinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236).The speed of quantitation of multiple samples may be accelerated byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or colorimetricresponse gives rapid quantitation.

In further embodiments, an oligonucleotide derived from any of thepolynucleotide sequences described herein may be used as a target in amicroarray. The microarray can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information will be useful in determining gene function,understanding the genetic basis of disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents (Heller, R.et al. (1997) Proc. Natl. Acad. Sci. 94:2150-55).

In one embodiment, the microarray is prepared and used according to themethods described in PCT application WO95/11995 (Chee et al.), Lockhart,D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al.(1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which areincorporated herein in their entirety by reference.

The microarray is preferably composed of a large number of unique,single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably 15-30 nucleotides in length, and most preferablyabout 20-25 nucleotides in length. For a certain type of microarray, itmay be preferable to use oligonucleotides which are only 7-10nucleotides in length. The microarray may contain oligonucleotides whichcover the known 5', or 3', sequence, sequential oligonucleotides whichcover the full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of the sequence is knownor that are specific to one or more unidentified cDNAs which are commonto a particular cell type, developmental or disease state.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5' or more preferably at the 3' end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In certain situations it may beappropriate to use pairs of oligonucleotides on a microarray. The"pairs" will be identical, except for one nucleotide which preferably islocated in the center of the sequence. The second oligonucleotide in thepair (mismatched by one) serves as a control. The number ofoligonucleotide pairs may range from two to one million. The oligomersare synthesized at designated areas on a substrate using alight-directed chemical process. The substrate may be paper, nylon orother type of membrane, filter, chip, glass slide or any other suitablesolid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a "gridded" array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray, the RNA or DNAfrom a biological sample is made into hybridization probes. The mRNA isisolated, and cDNA is produced and used as a template to make antisenseRNA (aRNA). The aRNA is amplified in the presence of fluorescentnucleotides, and labeled probes are incubated with the microarray sothat the probe sequences hybridize to complementary oligonucleotides ofthe microarray. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray. The biological samples may be obtained from any bodilyfluids (such as blood, urine, saliva, phlegm, gastric juices, etc.),cultured cells, biopsies, or other tissue preparations. A detectionsystem may be used to measure the absence, presence, and amount ofhybridization for all of the distinct sequences simultaneously. Thisdata may be used for large scale correlation studies on the sequences,mutations, variants, or polymorphisms among samples.

In another embodiment of the invention, the nucleic acid sequences whichencode MHCP may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome, to a specific regionof a chromosome or to artificial chromosome constructions, such as humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions orsingle chromosome cDNA libraries as reviewed in Price, C. M. (1993)Blood Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.

Fluorescent in situ hybridization (FISH as described in Verma et al.(1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York, N.Y.) may be correlated with other physical chromosome mappingtechniques and genetic map data. Examples of genetic map data can befound in various scientific journals or at Online Mendelian Inheritancein Man (OMIM). Correlation between the location of the gene encodingMHCP on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, MHCP, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenMHCP and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to MHCP large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with MHCP, or fragments thereof, and washed. Bound MHCP is thendetected by methods well known in the art. Purified MHCP can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding MHCP specificallycompete with a test compound for binding MHCP. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with MHCP.

In additional embodiments, the nucleotide sequences which encode MHCPmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES I cDNA Library Construction

LUNGNOT18

The LUNGNOT18 cDNA library was constructed from microscopically normalleft upper lobe lung tissue removed from a 66-year-old Caucasian femaleduring a segmental lung resection following a diagnosis of malignantlung neoplasm. Pathology for the associated tissue from the left upperlobe lung showed grade 2 adenocarcinoma with bronchoalveolar featuresand prominent inflammation forming a well-circumscribed nodular mass.The tumor did not involve the pleura. The bronchial margin was negativefor tumor. Five intrapulmonary peribronchial lymph nodes, inferiormediastinal lymph nodes comprising 6 subcarinal lymph nodes, 2 leftpulmonary artery lymph nodes, 2 left pulmonary artery lymph nodes, 4left paratracheal lymph nodes, and 5 left bronchial lymph nodes were allnegative for tumor. Patient history included cerebrovascular disease,atherosclerosis, pulmonary insufficiency, and a normal delivery.Previous surgery included endarterectomy. Patient medications includedTrental® (pentoxifylline; Hoechst-Roussel Pharmaceuticals, Inc.,Somerville, N.J.); Zocor; and EC-ASA. Family history included acutemyocardial infarction in the mother and father and atherosclerosis inthe sibling.

The frozen tissue was homogenized and lysed using a BrinkmannHomogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, N.J.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using a Beckman SW28 rotor in a Beckman L8-70MUltracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with phenol, pH 8.0,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water, and treated with DNase at 37° C.Extraction and precipitation were repeated as before. The mRNA was thenisolated using the Qiagen OLIGOTEX kit (QIAGEN, Inc., Chatsworth,Calif.) and used to construct the cDNA library.

The RNA was handled according to the recommended protocols in theSUPERSCRIPT plasmid system (Catalog #18248-013; GIBCO-BRL). cDNAsynthesis was initiated with a NotI-oligo d(T) primer. Double-strandedcDNA was blunted, ligated to EcoRI adaptors, digested with NotI,fractionated on a SEPHAROSE CL4B column (Catalog #275105, Pharmacia),and those cDNAs exceeding 400 bp were ligated into the NotI and EcoRIsites of the pINCY vector (Incyte pharmaceuticals, Palo Alto, Calif.).The plasmid pINCY was subsequently transformed into DH12S competentcells (Cat. #18312-017, GIBCO-BRL).

PENCNOT06

The PENCNOT06 cDNA library was constructed from penile corpora cavernosatissue obtained from an African American male (specimen #1011B).Pathology indicated surgical margins were free of neoplasm. Pathologyfor the associated tumor tissue indicated invasive grade 4 (of 4)urothelial carcinoma forming a soft tissue scrotal mass that invaded thecavernous body of the penis and encased both testicles. The rightinguinal lymph node showed metastatic grade 4 (of 4) urothelialcarcinoma, with extranodal invasion.

The frozen tissue was homogenized and lysed in TRIZOL reagent (1 gtissue/10 ml TRIZOL; Cat. #10296-028; GIBCO-BRL), a monoplastic solutionof phenol and guanidine isothiocyanate, using a Brinkmann HomogenizerPolytron PT-3000 (Brinkmann Instruments, Westbury, N.Y.). After a briefincubation on ice, chloroform was added (1:5 v/v) and the lysate wascentrifuged. The upper chloroform layer was removed to a fresh tube andthe RNA extracted with isopropanol, resuspended in DEPC-treated water,and DNase treated for 25 min at 37° C. Extraction and precipitation wererepeated as before. The mRNA was then isolated using the Qiagen OLIGOTEXkit (QIAGEN, Inc., Chatsworth, Calif.) and used to construct the cDNAlibrary.

The mRNA was handled according to the recommended protocols in theSUPERSCRIPT plasmid system (Cat. #18248-013, GIBCO-BRL). cDNA synthesiswas initiated with a NotI-oligo d(T) primer. Double-stranded cDNA wasblunted, ligated to EcoRI adaptors, digested with NotI, fractionated ona SEPHAROSE CL4B column (Cat. #275105-01; Pharmacia), and those cDNAsexceeding 400 bp were ligated into the NotI and EcoRI sites of the pINCYvector (Incyte). The plasmid pINCY was subsequently transformed intoDH5α competent cells (Cat. #18258-012; GIBCO-BRL).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA from both libraries were released from the cells andpurified using the REAL Prep 96 plasmid kit (Catalog #26173, QIAGEN,Inc.). The recommended protocol was employed except for the followingchanges: 1) the bacteria were cultured in 1 ml of sterile Terrific Broth(Catalog #22711, GIBCO-BRL) with carbenicillin at 25 mg/L and glycerolat 0.4%; 2) after inoculation, the cultures were incubated for 19 hoursand at the end of incubation, the cells were lysed with 0.3 ml of lysisbuffer; and 3) following isopropanol precipitation, the plasmid DNApellet was resuspended in 0.1 ml of distilled water. After the last stepin the protocol, samples were transferred to a 96-well block for storageat 4° C.

The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol.Biol. 94:441f), using a Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.)in combination with Peltier Thermal Cyclers (PTC200 from MJ Research,Watertown, Mass.) and Applied Biosystems 377 DNA Sequencing Systems; andthe reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences and/or amino acid sequences of the SequenceListing were used to query sequences in the GenBank, SwissProt, BLOCKS,and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofhomology using BLAST (Basic Local Alignment Search Tool; Altschul, S. F.(1993) J. Mol. Evol 36:290-300; Altschul, S. F. et al. (1990) J. Mol.Biol. 215:403-410).

BLAST produced alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST was especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal, or plant) origin. Other algorithms such asthe one described in Smith, T. et al. (1992, Protein Engineering5:35-51), incorporated herein by reference, could have been used whendealing with primary sequence patterns and secondary structure gappenalties. The sequences disclosed in this application have lengths ofat least 49 nucleotides, and no more than 12% uncalled bases (where N isrecorded rather than A, C, G, or T).

The BLAST approach searched for matches between a query sequence and adatabase sequence. BLAST evaluated the statistical significance of anymatches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻¹⁴ for peptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and other mammalian sequences (mam),and deduced amino acid sequences from the same clones were then searchedagainst GenBank functional protein databases (mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp)) for homology.

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. (1993)J.Mol.Evol. 36:290-300; Altschul, S. F. et al. (1990) J.Mol.Evol.215:403-410) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding MHCP occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of MHCP Encoding Polynucleotides

The nucleic acid sequences of the Incyte Clones 2220246 and 3440902 wereused to design oligonucleotide primers for extending a partialnucleotide sequence to full length. One primer was synthesized toinitiate extension in the antisense direction, and the other wassynthesized to extend sequence in the sense direction. Primers were usedto facilitate the extension of the known sequence "outward" generatingamplicons containing new, unknown nucleotide sequence for the region ofinterest. The initial primers were designed from the cDNA using OLIGO4.06 (National Biosciences), or another appropriate program, to be about22 to about 30 nucleotides in length, to have a GC content of 50% ormore, and to anneal to the target sequence at temperatures of about 68°to about 72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

Selected human cDNA libraries (Gibco/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. Beginning with 40 pmol of each primer and the recommendedconcentrations of all other components of the kit, PCR was performedusing the Peltier Thermal Cycler (PTC200; M. J. Research, Watertown,Mass.) and the following parameters:

    ______________________________________                                        Step 1       94° C. for 1 min (initial denaturation)                                 Step 2 65° C. for 1 min                                    Step 3 68° C. for 6 min                                                Step 4 94° C. for 15 sec                                               Step 5 65° C. for 1 min                                                Step 6 68° C. for 7 min                                                Step 7 Repeat step 4-6 for 15 additional cycles                               Step 8 94° C. for 15 sec                                               Step 9 65° C. for 1 min                                                Step 10 68° C. for 7:15 min                                            Step 11 Repeat step 8-10 for 12 cycles                                        Step 12 72° C. for 8 min                                               Step 13 4° C. (and holding)                                          ______________________________________                                    

A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were excised from the gel,purified using QIAQUICK (QIAGEN Inc., Chatsworth, Calif.), and trimmedof overhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2× Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2× Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

    ______________________________________                                        Step 1     94° C. for 60 sec                                             Step 2 94° C. for 20 sec                                               Step 3 55° C. for 30 sec                                               Step 4 72° C. for 90 sec                                               Step 5 Repeat steps 2-4 for an additional 29 cycles                           Step 6 72° C. for 180 sec                                              Step 7 4° C. (and holding)                                           ______________________________________                                    

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4 isused to obtain 5' regulatory sequences using the procedure above,oligonucleotides designed for 5' extension, and an appropriate genomiclibrary.

VI Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 or SEQ ID NO:4 areemployed to screen cDNAs, genomic DNAs, or mRNAs. Although the labelingof oligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³² P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN, Boston, Mass.). Thelabeled oligonucleotides are substantially purified with SEPHADEX G-25superfine resin column (Pharmacia & Upjohn). A aliquot containing 10⁷counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1,or Pvu II; DuPont NEN).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

VII Microarrays

To produce oligonucleotides for a microarray, the nucleotide sequencedescribed herein is examined using a computer algorithm which starts atthe 3' end of the nucleotide sequence. The algorithm identifiesoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that would interfere with hybridization. Thealgorithm identifies 20 sequence-specific oligonucleotides of 20nucleotides in length (20-mers). A matched set of oligonucleotides iscreated in which one nucleotide in the center of each sequence isaltered. This process is repeated for each gene in the microarray, anddouble sets of twenty 20 mers are synthesized and arranged on thesurface of the silicon chip using a light-directed chemical process(Chee, M. et al., PCT/WO95/11995, incorporated herein by reference).

In the alternative, a chemical coupling procedure and an ink jet deviceare used to synthesize oligomers on the surface of a substrate(Baldeschweiler, J. D. et al., PCT/WO95/25116, incorporated herein byreference). In another alternative, a "gridded" array analogous to a dot(or slot) blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available materials and machines and containgrids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.After hybridization, the microarray is washed to remove nonhybridizedprobes, and a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the micro-array.

VIII Complementary Polynucleotides

Sequence complementary to the MHCP-encoding sequence, or any partthereof, is used to decrease or inhibit expression of naturallyoccurring MHCP. Although use of oligonucleotides comprising from about15 to about 30 base-pairs is described, essentially the same procedureis used with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using Oligo 4.06 software and the codingsequence of MHCP, SEQ ID NO:1 or SEQ ID NO:3. To inhibit transcription,a complementary oligonucleotide is designed from the most unique 5'sequence and used to prevent promoter binding to the coding sequence. Toinhibit translation, a complementary oligonucleotide is designed toprevent ribosomal binding to the MHCP-encoding transcript.

IX Expression of MHCP

Expression of MHCP is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express MHCP in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion ofMHCP into the bacterial growth media which can be used directly in thefollowing assay for activity.

X Demonstration of MHCP Activity

The assay for myosin heavy chain-like proteins is based upon the abilityof MHCPs to interact with actinomyosin filaments in vitro (Ho, G. andChisholm, R. L. (1997) J. Biol. Chem. 272:4522-4527). Actin-activatedATPase is assayed in buffer A (10 mM Tris-HCl, pH 7.6, 25 mM KCl, 5 mMMgCl₂, 0.1 mM CaCl₂, 1 mM ATP), 0-10 μM MHCP, 0-10 μM actin, and 50μg/ml myosin. Ca²⁺ -activated ATPase is assayed in buffer B (20 mMTris-HCl, pH 8.0, 500 mM KCl, 10 mM CaCl₂, 1 mM ATP), 0-10 μM MHCP, and50 μg/ml myosin. Reactions are incubated at room temperature for 5 min,quenched with acid, and the liberated inorganic phosphate (P_(i)) isquantified following organic extraction.

In vitro motility assays are performed as follows. Myosin is diluted to200 μg/ml in buffer C (25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl₂, 1mM EGTA, 10 mM dithiothreitol), applied to a flow cell coated withnitrocellulose, and blocked with buffer C containing 0.5 mg/ml BSA(C/BSA). A solution of phalloidin-labeled actin is perfused followed by1 mM ATP in C/BSA to remove myosin heads that bind actin in a rigorfashion. After washing with C/BSA to remove the excess nonfluorescentactin, a solution of rhodamine-phalloidin-labeled actin and MHCP inC/BSA is introduced. Active movement is initiated at room temperature byintroducing C/BSA containing 1 mM ATP and oxygen scavenger enzymes.Images (recorded using a Zeiss standard microscope (Zeiss, N.Y.)equipped with a Hamamatsu SIT camera (Hamamatsu, Japan)) of movingmyotubes are tracked for up to 30s, and translocation velocitiescalculated using the myotube centroids to establish initial and finalpositions for 2s or 4s samples during the continuous movement.

XI Production of MHCP Specific Antibodies

MHCP that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 or SEQ ID NO:4 is analyzed usingLASERGENE software (DNASTAR Inc) to determine regions of highimmunogenicity and a corresponding oligopeptide is synthesized and usedto raise antibodies by means known to those of skill in the art.Selection of appropriate epitopes, such as those near the C-terminus orin hydrophilic regions, is described by Ausubel et al. (supra), andothers.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio iodinated, goat anti-rabbitIgG.

XII Purification of Naturally Occurring MHCP Using Specific Antibodies

Naturally occurring or recombinant MHCP is substantially purified byimmunoaffinity chromatography using antibodies specific for MHCP. Animmunoaffinity column is constructed by covalently coupling MHCPantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing MHCP is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof MHCP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/MHCP binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope, such as urea or thiocyanate ion), and MHCP is collected.

XIII Identification of Molecules Which Interact with MHCP

MHCP or biologically active fragments thereof are labeled with ¹²⁵ IBolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled MHCP, washed and any wells withlabeled MHCP complex are assayed. Data obtained using differentconcentrations of MHCP are used to calculate values for the number,affinity, and association of MHCP with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 5                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 226 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: LUNGNOT18                                                        (B) CLONE: 2220246                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  Met Tyr Cys Cys Arg Val Thr Ser Gln Ser - #Leu Gln Leu Pro Tyr        Gly                                                                               1               5 - #                 10 - #                 15             - -  Pro Phe Val Met Val Gly Phe Ser Pro Leu - #Gln Lys His Gly Leu Val                   20     - #             25     - #             30                  - -  Ile Ile Pro Asp Gly Thr Pro Asn Gly Asp - #Val Ser His Glu Pro Val               35         - #         40         - #         45                      - -  Ala Gly Ala Ile Thr Val Val Ser Gln Glu - #Ala Ala Gln Val Leu Glu           50             - #     55             - #     60                          - -  Ser Ala Gly Glu Gly Pro Leu Asp Val Arg - #Leu Arg Lys Leu Ala Gly       65                 - # 70                 - # 75                 - # 80       - -  Glu Lys Glu Glu Leu Leu Ser Gln Ile Arg - #Lys Leu Lys Leu Gln Leu                       85 - #                 90 - #                 95              - -  Glu Glu Glu Arg Gln Lys Cys Ser Arg Asn - #Asp Gly Thr Val Gly Asp                   100     - #            105     - #            110                 - -  Leu Ala Gly Leu Gln Asn Gly Ser Asp Leu - #Gln Phe Ile Glu Met Gln               115         - #        120         - #        125                     - -  Arg Asp Ala Asn Arg Gln Ile Ser Glu Tyr - #Lys Phe Lys Leu Ser Lys           130             - #    135             - #    140                         - -  Ala Glu Gln Asp Ile Thr Thr Leu Glu Gln - #Ser Ile Ser Arg Leu Glu       145                 - #150                 - #155                 -         #160                                                                             - -  Gly Gln Val Leu Arg Tyr Lys Thr Ala Ala - #Glu Asn Ala Glu Lys        Val                                                                                              165 - #                170 - #                175            - -  Glu Asp Glu Leu Lys Ala Glu Lys Arg Lys - #Leu Gln Arg Glu Leu Arg                   180     - #            185     - #            190                 - -  Thr Ala Leu Asp Lys Ile Glu Glu Met Glu - #Met Thr Asn Ser His Leu               195         - #        200         - #        205                     - -  Ala Lys Arg Leu Glu Lys Met Lys Ala Asn - #Arg Thr Ala Leu Leu Ala           210             - #    215             - #    220                         - -  Gln Gln                                                                  225                                                                           - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2102 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: LUNGNOT18                                                        (B) CLONE: 2220246                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -  GTACTTCTCT GCTACCTTTC ACCTGCTCTT TTTCTGTGCC ATTGTTTCCC - #TTGCTACCT    T    60                                                                         - -  ACCTAAAACC AGGCCCTAGA GAAACAGAAA GAATACATTG CCTGCCTTAG - #GAATGAGCG    A   120                                                                         - -  GATATGCTCA GAGAGGAGCT GGCTGACCTG CAGGAGACAG TGAAGACGGG - #AGAGGTATG    T   180                                                                         - -  TAGCATTAGC CTGGAATTCA GGTCCCTCAC TGTTTTACTC TCTATCTTCC - #TTCCTTTCA    T   240                                                                         - -  CCTGCCATCT TTCCTAGCCT AAATACAAAC TACAGTGTTT ATTCTCTAAT - #CCAGATTTG    G   300                                                                         - -  TAGGTTGAAG CTATTTCTTA CACAGAGCTA TATTTCATGT AACTGATTCT - #AACCAGGTT    T   360                                                                         - -  TACCTGTAGC AAACATGTAT TGTTGCAGAG TGACCTCACA GAGCTTACAG - #CTTCCATAT    G   420                                                                         - -  GGCCTTTTGT GATGGTGGGT TTTTCCCCCC TGCAGAAACA TGGCTTAGTT - #ATAATCCCC    G   480                                                                         - -  ATGGCACTCC CAATGGTGAT GTCAGTCATG AACCAGTGGC TGGAGCCATC - #ACTGTTGTG    T   540                                                                         - -  CTCAGGAAGC TGCTCAGGTC TTGGAGTCAG CAGGAGAAGG GCCATTAGAT - #GTAAGGCTA    C   600                                                                         - -  GAAAACTTGC TGGAGAGAAG GAAGAACTAC TGTCACAGAT TAGAAAACTG - #AAGCTTCAG    T   660                                                                         - -  TAGAGGAGGA ACGACAGAAA TGCTCCAGGA ATGATGGCAC AGTGGGTGAC - #CTGGCAGGA    C   720                                                                         - -  TGCAGAATGG CTCAGACTTG CAGTTCATCG AAATGCAGAG AGATGCCAAT - #AGACAAATT    A   780                                                                         - -  GCGAATACAA ATTTAAGCTT TCAAAAGCAG AACAGGATAT AACTACCTTG - #GAGCAAAGT    A   840                                                                         - -  TTAGCCGGCT TGAGGGACAG GTTCTGAGAT ATAAAACTGC TGCTGAGAAT - #GCTGAGAAA    G   900                                                                         - -  TTGAAGATGA ATTGAAAGCA GAAAAACGGA AGCTACAACG AGAGTTACGA - #ACAGCACTG    G   960                                                                         - -  ACAAGATTGA GGAGATGGAG ATGACCAACA GCCACCTGGC CAAGCGGCTG - #GAGAAGATG    A  1020                                                                         - -  AGGCCAATAG GACAGCACTT CTGGCCCAGC AGTAGGAAAA CCACCCTTCA - #ACCTGGGTG    A  1080                                                                         - -  TGCTCCTTGG GGCCCTACCT AGAGGGACTG ACTTTTGTCC ATTGACACAA - #ACCCCTTTT    A  1140                                                                         - -  GTACTGTTTT GAGTTTTGTC ATTAAAACAG CCACCTTTGT ATTTTATAAT - #TTATGACAG    A  1200                                                                         - -  ATGAAGTCAT TTTGAATCTA CATGAATGAA CACTTTGGAT TTTGTTGTAG - #TTTGATTCT    A  1260                                                                         - -  GGGTAGAACC AGTCCATGCT GTTTTTATTT TTTATCTCCG TAATTGTAGA - #ATCATGTTT    A  1320                                                                         - -  CTCAACGTTT TTCCCCAGCT GCCTCAGTAA CTGGGCACTC GGAGGCCTTG - #GCACGGGTT    C  1380                                                                         - -  TGGAGGACAG ACAGCAATTC TATGAGTGCT CACTGAGATA CTTGCTGGAG - #ACCTCAGAA    A  1440                                                                         - -  ACACAAGTGC CTTCTCCACG GTGCAATTCA GACTTCAGTG ATCTCCAGTG - #GTCAAAAGA    C  1500                                                                         - -  ATTTACCCTT AATATCAGAC AACATTTATA TTTTAGTGAA GAAACAAGTT - #CTCGGGTGG    G  1560                                                                         - -  GAATCTATGT TTCACTCAGA TTTATATGTT TGGAGGAAAA AAGCCTTTTT - #TTGTAAAAT    A  1620                                                                         - -  TTTAAATTTA TATAAGAAAA TGTTAGAAAA AAATATGGGG AGTGTATATA - #AAACTTGCT    T  1680                                                                         - -  TATTGCATGG GGCAGGGGAA GTCCAGGCCT AATACTCCTA AAGTAAGAGT - #TGGGTCCTT    T  1740                                                                         - -  TTTTCTTCAA TACAACTGTG CTGTACCTTG TAAAGTATTT TATCTGCTGC - #TTATTTGTG    G  1800                                                                         - -  AATGAAACCT CAAACAAACC CAAAGGGGGA GGGTAGGGCA GGGCAGGCAG - #ATTGGAAAT    C  1860                                                                         - -  TGCCTGCAGA TTCTATTAAA TACACCCTTT TGCCAACCAA AAAAAAAAAG - #GTTAAAAAA    G  1920                                                                         - -  GCGAAACAGG GTGGTCTGTA TAGGGACAGG AAAGGAAAAA AAAAAAGGGG - #GGGCCCCCG    A  1980                                                                         - -  ATTTTGGGAC CCCTCGCCCC GGGGAATTAT TTCCGGGCCG GGTTCCTGGA - #GGGGTACCA    T  2040                                                                         - -  TTTTCCCTAA AAGGGAGGCC GTTTTAACCG CCTGGGGGTA ATCCAGGGCC - #AAGGTGTTT    T  2100                                                                         - -  TC                 - #                  - #                  - #                2102                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 353 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: PENCNOT06                                                        (B) CLONE: 3440902                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -  Met Lys Glu Leu Glu Arg Gln Gln Lys Glu - #Val Glu Glu Arg Pro Glu        1               5 - #                 10 - #                 15              - -  Lys Asp Phe Thr Glu Lys Gly Ser Arg Asn - #Met Pro Gly Leu Ser Ala                   20     - #             25     - #             30                  - -  Ala Thr Leu Ala Ser Leu Gly Gly Thr Ser - #Ser Arg Arg Gly Ser Gly               35         - #         40         - #         45                      - -  Asp Thr Ser Ile Ser Ile Asp Thr Glu Ala - #Ser Ile Arg Glu Ile Lys           50             - #     55             - #     60                          - -  Asp Ser Leu Ala Glu Val Glu Glu Lys Tyr - #Lys Lys Ala Met Val Ser       65                 - # 70                 - # 75                 - # 80       - -  Asn Ala Gln Leu Asp Asn Glu Lys Thr Asn - #Phe Met Tyr Gln Val Asp                       85 - #                 90 - #                 95              - -  Thr Leu Lys Asp Met Leu Leu Glu Leu Glu - #Glu Gln Leu Ala Glu Ser                   100     - #            105     - #            110                 - -  Arg Arg Gln Tyr Glu Glu Lys Asn Lys Glu - #Phe Glu Arg Glu Lys His               115         - #        120         - #        125                     - -  Ala His Ser Ile Leu Gln Phe Gln Phe Ala - #Glu Val Lys Glu Ala Leu           130             - #    135             - #    140                         - -  Lys Gln Arg Glu Glu Met Leu Glu Lys His - #Gly Ile Ile Leu Asn Ser       145                 - #150                 - #155                 -         #160                                                                             - -  Glu Ile Ala Thr Asn Gly Glu Thr Ser Asp - #Thr Leu Asn Asn Val        Gly                                                                                              165 - #                170 - #                175            - -  Tyr Gln Gly Pro Thr Lys Met Thr Lys Glu - #Glu Leu Asn Ala Leu Lys                   180     - #            185     - #            190                 - -  Ser Thr Gly Asp Gly Thr Leu Asp Ile Arg - #Leu Lys Lys Leu Val Asp               195         - #        200         - #        205                     - -  Glu Arg Glu Cys Leu Leu Glu Gln Ile Lys - #Lys Leu Lys Gly Gln Leu           210             - #    215             - #    220                         - -  Glu Glu Arg Gln Lys Ile Gly Lys Leu Asp - #Asn Leu Arg Ser Glu Asp       225                 - #230                 - #235                 -         #240                                                                             - -  Asp Val Leu Glu Asn Gly Thr Asp Met His - #Val Met Asp Leu Gln        Arg                                                                                              245 - #                250 - #                255            - -  Asp Ala Asn Arg Gln Ile Ser Asp Leu Lys - #Phe Lys Leu Ala Lys Ser                   260     - #            265     - #            270                 - -  Glu Gln Glu Ile Thr Ala Leu Glu Gln Asn - #Val Ile Arg Leu Glu Ser               275         - #        280         - #        285                     - -  Gln Val Ser Arg Tyr Lys Ser Ala Ala Glu - #Asn Ala Glu Lys Ile Glu           290             - #    295             - #    300                         - -  Asp Glu Leu Lys Ala Glu Lys Arg Lys Leu - #Gln Arg Glu Leu Arg Ser       305                 - #310                 - #315                 -         #320                                                                             - -  Ala Leu Asp Lys Thr Glu Glu Leu Glu Val - #Ser Asn Gly His Leu        Val                                                                                              325 - #                330 - #                335            - -  Lys Arg Leu Glu Lys Met Lys Ala Asn Arg - #Ser Ala Leu Leu Ser Gln                   340     - #            345     - #            350                 - -  Gln                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1969 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: PENCNOT06                                                        (B) CLONE: 3440902                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -  AAAGCCGGGA GATCGACTGT TTGAGCCCGG AAGCGCAGAA GCTGGCGGAA - #GCCCGGCTC    G    60                                                                         - -  CTGCAAAACG GGCGGCCCGC GCGGAGGCTC GCGAGATCCG CATGAAGGAG - #CTGGAGCGG    C   120                                                                         - -  AGCAGAAGGA GGTAGAAGAG AGACCAGAAA AAGATTTTAC TGAGAAGGGG - #TCTCGTAAC    A   180                                                                         - -  TGCCGGGCCT GTCTGCAGCC ACGCTGGCCT CTCTGGGTGG GACTTCCTCT - #CGGAGAGGC    A   240                                                                         - -  GCGGAGACAC CTCCATCTCC ATCGACACCG AGGCATCCAT CAGGGAAATC - #AAGGACTCT    C   300                                                                         - -  TAGCAGAAGT TGAAGAGAAA TATAAGAAGG CTATGGTTTC CAATGCTCAG - #CTAGACAAT    G   360                                                                         - -  AAAAGACAAA CTTCATGTAC CAGGTTGATA CCCTAAAAGA TATGTTGCTG - #GAGCTTGAA    G   420                                                                         - -  AACAGCTGGC TGAATCTAGG CGGCAGTACG AAGAGAAAAA CAAAGAATTT - #GAAAGGGAA    A   480                                                                         - -  AACACGCCCA CAGTATACTG CAATTTCAGT TTGCTGAAGT CAAGGAGGCC - #CTGAAGCAA    A   540                                                                         - -  GAGAGGAAAT GCTCGAGAAA CATGGAATAA TCCTAAATTC AGAAATAGCT - #ACCAATGGA    G   600                                                                         - -  AGACTTCCGA CACCCTCAAT AATGTTGGAT ACCAAGGTCC TACCAAGATG - #ACAAAAGAA    G   660                                                                         - -  AGTTAAATGC CCTCAAGTCG ACAGGGGATG GGACCCTAGA TATTAGGTTG - #AAAAAGCTG    G   720                                                                         - -  TTGATGAACG GGAATGCTTA TTGGAACAGA TTAAGAAACT CAAAGGGCAG - #CTGGAGGAG    A   780                                                                         - -  GACAGAAGAT TGGCAAACTA GACAATCTTC GATCTGAAGA TGATGTCTTG - #GAAAACGGG    A   840                                                                         - -  CAGACATGCA TGTAATGGAC CTACAAAGGG ATGCCAACAG ACAGATCAGC - #GACCTCAAA    T   900                                                                         - -  TTAAACTTGC AAAATCTGAG CAAGAGATAA CTGCATTAGA ACAAAATGTA - #ATAAGGTTA    G   960                                                                         - -  AGAGTCAAGT ATCACGTTAC AAATCAGCGG CTGAAAATGC AGAAAAAATA - #GAAGATGAA    C  1020                                                                         - -  TTAAGGCAGA AAAACGGAAA CTCCAAAGAG AGCTCCGCTC TGCATTGGAT - #AAAACAGAA    G  1080                                                                         - -  AGCTCGAGGT GAGCAACGGC CACTTAGTGA AGCGTCTGGA AAAAATGAAA - #GCAAATCGG    A  1140                                                                         - -  GTGCACTCTT GTCCCAGCAG TAAATTCCAG CTCTGATCAG GCAACTGGTT - #GGTGACTGG    A  1200                                                                         - -  GAGCATTGTT TCATAGGCTT TTCTCTGTCC TGTCTGGGAG CGCTGCTTCT - #TCCCCTGCC    T  1260                                                                         - -  TCTGAGAGAC GAAGACCGTG GCGAGCTTGG CGCTTAGGGG CTCCCGTGCC - #ATGGCTCAC    C  1320                                                                         - -  CCAGGGAGCC CCAGCAGCCA CCAGGTGCCT CTGTCTGCAG ACCCCTGGCC - #CGGGCTGGC    G  1380                                                                         - -  CCGACGCTCA GAACCTGCAG GTACTTCATA AGCACACAGG GGCCTCGAGG - #GAGCTCTGT    G  1440                                                                         - -  TCTGACCGCA CAGCAGCCTC TGAATGCCGC TGGAAGTGAT GATCAAAGTA - #AAGATTCAG    T  1500                                                                         - -  TGGGACTTGA GTTTTTTTTT TTTTCATGTG TCTTGCTGAA GATTAAGGGG - #AAATGTTAC    A  1560                                                                         - -  GTGTTGGGAC TTCCTTTCAT GGCAGAATCT ACAATTTGAG CGACTTCAGT - #AGTATCTCT    T  1620                                                                         - -  AGTCTACGCT TTTCATACAC AAAACACTGT GGAACCACAA GCCATTACCA - #AGCAAAACT    C  1680                                                                         - -  TTTCACTGGA AACAAGGGGG CAGTCTAGAA GTAAAAGTGA CCTTAAGAAG - #ACTCTTTAC    A  1740                                                                         - -  GGCAACAAAT GAAGCTTTTC TAAGGGATTT TTGCATCAGT TCAGTCATAA - #GAATACTTT    T  1800                                                                         - -  TTCCAGGGTA ATTAGGCAAT AGCTTCACTG AAAATGACAG CTTTTCATTG - #CATTATTTA    A  1860                                                                         - -  TCCTTATATT TGGAATTGAA GTCGTTAACT TCTTTTAAAG AATGTACTAT - #TAGAAAAAT    T  1920                                                                         - -  AAAAATGAAA TGTTGAGAGA CTTCAAAAAA AAAAAAAAAA AAAAAAAAA - #                 1969                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 133 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 532473                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - -  Met Arg Lys Asp Tyr Glu Glu Leu Glu Gln - #Thr Ile Tyr Thr Gln Arg        1               5 - #                 10 - #                 15              - -  His Ala Arg Asn Ala Arg Asp Ser Ala Asn - #Val Ile Pro Asn Gln Gly                   20     - #             25     - #             30                  - -  Val Asp Asp Val Asn Lys Asp Ala Ala Lys - #Gln Leu Ala Glu Met Lys               35         - #         40         - #         45                      - -  Leu Lys Met Gln Asp Leu Glu Arg Glu Asn - #Thr Asn Gln Gln Gly Asn           50             - #     55             - #     60                          - -  Val Ile Arg Met Glu Gly Gln Met Lys Arg - #Tyr Lys Ser Asn Ala Asp       65                 - # 70                 - # 75                 - # 80       - -  Val Ala Glu Lys Glu Leu Asp Glu Leu Lys - #Thr Gln Met Arg Gln Thr                       85 - #                 90 - #                 95              - -  Lys Lys Glu Leu Arg Asp Lys Glu Asn Ala - #Leu Asp Glu Gln Lys Glu                   100     - #            105     - #            110                 - -  Thr Asn Lys His Leu Gln Ser Arg Leu Glu - #Lys Met Arg Met Gln Arg               115         - #        120         - #        125                     - -  Thr Gly Arg Pro Leu                                                          130                                                                     __________________________________________________________________________

What is claimed is:
 1. An isolated and purified polynucleotide sequenceencoding the myosin heavy chain-like protein having the amino acidsequence set forth in SEQ ID NO:1 or SEQ ID NO:3.
 2. A compositioncomprising the polynucleotide sequence of claim
 1. 3. An isolated andpurified polynucleotide sequence which is complementary to thepolynucleotide sequence of claim
 1. 4. An isolated and purifiedpolynucleotide sequence comprising the nucleic acid sequence set forthin SEQ ID NO:2 or SEQ ID NO:4.
 5. An isolated and purifiedpolynucleotide sequence which is complementary to the polynucleotidesequence of claim
 4. 6. An expression vector containing thepolynucleotide sequence of claim
 1. 7. A host cell containing theexpression vector of claim
 6. 8. A method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3, themethod comprising the steps of:a) culturing the host cell of claim 7under conditions suitable for the expression of the polypeptide; and b)recovering the polypeptide from the host cell culture.